The overall objectives of this program are to obtain information about: (1) Transport of folate compounds (including the cancer chemotherapeutic agent, Methotrexate (MTX) into L1210 and Lactobacillus casei cells; and the (2) Mechanism of folate-dependent enzymes such as dihydrofolate reductase, methionine synthetase and folinate isomerase. Projects to be undertaken under (1) include: (a) purification and characterization of the 5-methyltetrahydrofolate/MTX binding protein from L1210 cell membranes; (b) regulation of MTX transport by cyclic nucleotides; and (c) studies on the folate-binding protein from L. casei including its structure, behavior in Triton micelles, reconstitution in liposomes, energy-coupling, and high and low affinity forms. Under (2), the L1210 dihydrofolate reductase will be characterized with respect to (a) the nature and spatial relationship of the substrate-binding sites; and (b) the MTX-binding properties of naturally-occurring and synthetic multiple forms. Methionine synthetase will be purified, via affinity chromatography from bovine liver and L1210 cells, and the role of this enzyme (along with vitamin B12 and transcobalamin-II) in the replication of L1210 cells will be examined. The methionine synthetase system from Escherichia coli K-12 (B12-protein and two flavoproteins) will be further characterized with respect to: (a) sulfhydryl groups on the B12-protein; (b) reduction potential of the bound B12; (c) mechanisms of electron transfer; and (d) physical interaction of the three proteins. Folinate isomerase, purified from bovine liver or Micrococcus aerogenes, will be investigated with regard to the mechanism of ATP-dependent intermolecular transfer of the formyl group, and the level of the enzyme will be measured in various normal and tumor tissues.